The design, synthesis, and evaluation of an extensive series of CC-1065 and duocarmycin analogues are described in efforts to define fundamental relationships between structure, reactivity, and biological properties. These include deep-seated structural changes in the DNA alkylation subunits, the exploration of a novel series of alkylation subunit DNA crosslinking analogues, and the extensive examination of the DNA binding domain through application of solution-phase combinatorial chemistry techniques. Each of these modifications is made to address a specific question or concept relating structure and function (structure-reactivity or structure-activity). Central to these studies is the use of fundamental physical organic principles to define the recognition features responsible for the sequence selective DNA alkylation reaction and its source of catalysis. Through these studies, we hope to fully characterize the first well-defined example of a new mechanism of in situ activation of a DNA alkylating antitumor agent and the first example of activation at and by the biological target. [unreadable] [unreadable] The introduction and continued development of new techniques for establishing DNA binding selectivity and affinity promises to facilitate these and related studies. [unreadable] [unreadable]